The long term goal of this research is to discover and elucidate the series of molecular events that constitute the mechanism(s) of action of insulin. This proposal will exploit the insulin- stimulated phosphorylation of ribosomal protein S6 to dissect signaling mechanisms used by insulin. The properties of this response that make it logical-to use for this purpose are: 1) Its magnitude; insulin can increase the incorporation of labeled phosphate into S6 by 20-fold and the stoichiometry of phosphorylation from less than 1 up to 5 mol of phosphate per mol of S6. 2) Its stability; extracts from insulin-treated cells phosphorylate S6 up to 10-fold faster than extracts from untreated cells. Finally 3) the effect can be elicited by insulin in a cell lysate indicating a potential for reconstitution in vitro. Insulin-treated cells and tissues contain an insulin-stimulated protein kinase that can be purified in an activated form. The specific aims of this proposal focus on the isolation and study of this insulin-stimulated ribosomal protein S6 kinase as follows: 1) purify it; 2) make antibodies to it; 3) clone it; 4) isolate its inactive form; 5) examine the mechanism of its regulation; and 6) examine its role in insulin-stimulated S6 phosphorylation in intact cells. Since the activated form of this S6 protein kinase can be isolated, study of this enzyme offers a means of identifying the intermediates in the pathway of its activation. Elucidation of the pathway of regulation of this enzyme by insulin will enable reconstruction of the series of steps that constitute one mechanism of action of the hormone.